The long-term objective of this proposal is to understand the molecular and cellular mechanisms that generate calcium signals in T lymphocytes. The elevation of intracellular free Ca2+ concentration is a required signal that allows T cells to respond to foreign antigens by proliferating and acquiring functional abilities. The Ca2+ release-activated Ca2+ (CRAC) channel plays a central role in this process, by generating the sustained influx of Ca2+ necessary for induction of T cell activation genes. CRAC channels are the prototypic members of a ubiquitous family of store-operated channels that open in response to the depletion of the Ca2+ stored in the endoplasmic reticulum (ER). Despite more than 20 years of research, the mechanism that links store depletion to channel activation has remained a longstanding mystery. However, the recent identification of STIM1 as the ER Ca2+ sensor and Orai1 as a subunit of the CRAC channel has catalyzed rapid progress in elucidating the mechanism of CRAC channel activation. Store depletion causes STIM1 and Orai1 to redistribute within the ER and plasma membranes, respectively, so that they ultimately accumulate in regions (ER-PM junctions) where the ER is 10-25 nm from the PM. Their interaction at these sites leads to localized activation of CRAC channels and Ca2+ influx. Although we now have an outline of events underlying CRAC channel activation, there are many critical events that are not understood. The goal of this proposal is to elucidate in molecular detail the dynamic events leading from store depletion to the formation of the CRAC channel activation complex. Our recent studies suggest that multimerization of STIM1 is the key event that triggers CRAC channel activation. The first aim of this proposal is to determine the subunit stoichiometries of STIM1 and the CRAC channel before and after store depletion, using single-molecule photobleaching techniques in intact cells. Quantitative two-color imaging will be applied to measure the ratio of STIM1 and the CRAC channel as they co-assemble at ER-PM junctions, to provide the first quantitative description of the formation of the CRAC channel activation complex. The second aim is focused on the interaction of STIM1 and Orai1 and makes use of a fragment of STIM1 that we have found to activate CRAC channels strongly without supporting Ca2+-dependent inactivation. Through truncations and extensions of this domain we will identify the critical regions of STIM1 involved in activation and inactivation. The potency of this domain will be determined by applying purified protein to CRAC channels, and its ability to activate Orai1 through a direct interaction will be tested in a Ca2+ flux assay system reconstituted from purified proteins. The third aim applies single- molecule tracking to address whether STIM1 and Orai1 move by passive diffusion, and in combination with GFP photoactivation techniques, to reveal the turnover of these proteins within the activation complex. Together, the proposed experiments will provide a quantitative basis for understanding the critical events that link store depletion to CRAC channel activation. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the mechanism that controls calcium entry through ion channels in T lymphocytes. Mutations that inhibit this process lead to a lethal, severe combined immunodeficiency in human patients, showing that calcium entry is absolutely required for proper immune system function and survival. A better understanding of the molecular mechanism of calcium entry in T cells is important for identifying novel targets for drug development aimed at enhancing immune function in individuals with compromised immunity, or at inhibiting the immune response to treat autoimmune disease (e.g., diabetes, lupus, arthritis) or prevent the rejection of organ transplants.